The term "pneumocystis carinii" as used herein, refers to fungus classified as such by a method for phylogenetic grouping based upon the sequences of small subunit rRNA (Edman J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J. and Sogin, M. L. [1988] Nature, 334:519). Pneumocystis carinii is a member of the genus Pneumocystis. Pneumocystis carinii is an opportunistic lower respiratory tract pathogen. It is a small unicellular organism and is believed to exist in the mammalian host in two basic forms: the trophozoite and the cyst.
Pneumocystis carinii infects humans and most mammalian host. Although the parasite rarely causes illness in normal individuals, it characteristically gives rise to life threatening interstitial pneumonia in certain conditions of immunodeficiency and is one of the most common causes of morbidity and mortality in acquired immune deficiency syndrome.
Current means of diagnosis of Pneumocystis pneumonia include the use of histological techniques, such as Gomori's methenamine silver, Toludine Blue or Giemsa stains, on respiratory tract samples such as Bronchoalveolar lavage sample or, more commonly, induced sputum samples. These staining procedures require the specialist expertise of a cytologist to interpret the results. A number of rapid laboratory methods recently have become available. Some of them are antibody-based tests whereas others are DNA based tests. It is an aspect of the present invention to provide nucleic acid probes which are specific for Pneumocystis and which do not react with other fungi and bacteria which may be present in sampled materials. Such probes may be used in a variety of assay systems which avoid many of the disadvantages associated with the traditional methods.
It is another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make Pneumocystis carinii specific probes.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to procaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to fungus and in particular to human Pneumocystis carinii and do not provide Pneumocystis carinii specific probes useful in assays for detecting Pneumocystis carinii in clinical samples.
Hogan et al. (European patent publication WO 88/03957) describe a number of probes which are claimed to hybridize to a broad representation of eubacteria and fungi. However, Hogan et al. do not disclose the probes of the present invention, nor do they provide the teaching necessary to design such probes.
Edman, Kovacs, Masur, Santi, Elwood and Sogin (Nature, 334, pp. 519-522, 1988) describe a set of three probes to the 18S rRNA of the rat Pneumocystis. There is no known correlation between rat Pneumocystis and human Pneumocystis infections since it is not known whether rat Pneumocystis can cause the human disease. Edman et al. do not disclose the human Pneumocystis probes or the ferret Pneumocystis probes of the present invention, nor do they provide the teaching necessary to design such probes.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain four distinct RNA molecules which, at least in Saccharomyces cerevisiae, are referred to as 5S, 5.8S, 18S and 28S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 5.8S, 18S, and 28S rRNA are commonly used as generic names for the homologous RNA molecules in any eubacterial cell and this convention will be continued herein.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see below--Hybridization) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescien, 32-P, biotin, etc.), or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds.
The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.